U.S. patent application number 10/100697 was filed with the patent office on 2002-10-17 for aryl and heteroaryl fused aminoalkyl-imidazole derivatives: selective modulators of bradykinin b2 receptors.
This patent application is currently assigned to Neurogen Corporation. Invention is credited to Brielmann, Harry L., DeSimone, Robert W., Hutchison, Alan, Lew, Richard, Maynard, George D., Peterson, John M., Shaw, Kenneth.
Application Number | 20020151550 10/100697 |
Document ID | / |
Family ID | 26825698 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151550 |
Kind Code |
A1 |
DeSimone, Robert W. ; et
al. |
October 17, 2002 |
Aryl and heteroaryl fused aminoalkyl-imidazole derivatives:
selective modulators of bradykinin B2 receptors
Abstract
Disclosed are compounds of the formula: 1 or the
pharmaceutically acceptable non-toxic salts thereof wherein: A, B,
C. and D are N or CH; X is a bond or (un)substituted CH.sub.2;
R.sub.1 is lower alkenyl or (un)substituted lower alkyl; R.sub.3 is
lower alkyl; and R.sub.2, R.sub.4, R.sub.5, and R.sub.6 are
variables defined herein; which compounds are useful in the
diagnosis and treatment of renal diseases, heart failure,
hypertension, Meniere's disease, vaginal inflammation and pain,
peripheral circulatory disorders, climacteric disturbance,
retinochoroidal circulatroy disorders, myocardial ischemia,
myocardial infarction, postmyocardial infarction syndrome, angina
pectoris, restenosis after percutaneous transluminal coronary
angioplasty, hepatitis, liver cirrhosis, pancreatitis, ileus,
diabetes, diabetic complications, male infertility or glaucoma, or
for the increase of permeability of blood-brain barrier, pain,
asthma, and rhinitis.
Inventors: |
DeSimone, Robert W.;
(Durham, CT) ; Hutchison, Alan; (Madison, CT)
; Shaw, Kenneth; (Weston, CT) ; Maynard, George
D.; (Clinton, CT) ; Peterson, John M.;
(Madison, CT) ; Lew, Richard; (Hamden, CT)
; Brielmann, Harry L.; (Guilford, CT) |
Correspondence
Address: |
Steven J. Sarussi
McDonnell Boehnen Hulbert & Berghoff
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Assignee: |
Neurogen Corporation
Branford
CT
|
Family ID: |
26825698 |
Appl. No.: |
10/100697 |
Filed: |
March 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10100697 |
Mar 18, 2002 |
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09540580 |
Mar 31, 2000 |
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6358949 |
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60127505 |
Apr 2, 1999 |
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Current U.S.
Class: |
514/248 ;
514/263.1; 514/303; 514/394; 544/236; 544/277; 546/118;
548/309.7 |
Current CPC
Class: |
C07D 471/04
20130101 |
Class at
Publication: |
514/248 ;
514/263.1; 514/303; 514/394; 544/236; 544/277; 546/118;
548/309.7 |
International
Class: |
C07D 471/02; C07D
491/02; A61K 031/52 |
Claims
What is claimed is:
1. A compound of the formula: 23or pharmaceutically acceptable
salts thereof wherein: R.sub.1 is not 3-fluorobenzyl and represents
(i) (C.sub.2-C.sub.6)alkenyl; or (ii) R.sub.1 represents
aryl(C.sub.1-C.sub.6)alkyl or heteroaryl(C.sub.1-C.sub.6)alkyl,
where the ring portion of each is optionally substituted with one,
two or three groups independently selected from halogen, nitro,
trifluoromethyl, trifluoromethoxy, cyano, hydroxy,
(C.sub.1-C.sub.6)alkyl, hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono-
or di(C.sub.1-C.sub.6)alkylam- ino, amino (C.sub.1-C.sub.6) alkyl,
mono- or di (C.sub.1-C.sub.6)alkylamin- o(C.sub.1-C.sub.6)alkyl,
mono- or di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.- sub.6)alkoxy, or
(iii) OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.sub.8, O(CH.sub.2).sub.nCO.sub.2R.sub.7,
NR.sub.7COR.sub.8, COR.sub.7, CONR.sub.7R.sub.8 or CO.sub.2R.sub.7
where n=1, 2, 3, or 4; and R.sub.7 and R.sub.8 are the same or
different and represent hydrogen, SO.sub.2Me, or
(C.sub.1-C.sub.6)alkyl; or R.sub.7 and R.sub.8 together with the
nitrogen to which they are attached form a 5, 6 or 7 membered
carbocyclic ring where up to two of the members in the ring are
optionally hetero atoms selected from oxygen, sulfur and nitrogen,
and where each member is optionally substituted with
(C.sub.1-C.sub.6)alkyl; R.sub.2 represents hydrogen, hydroxy,
halogen, trifluoromethyl, trifluoromethoxy,
amino(C.sub.1-C.sub.6)alkyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6), or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4; and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring where up to two of the
members are optionally hetero atoms selected from oxygen, sulfur
and nitrogen, and where each member is optionally substituted with
(C.sub.1-C.sub.6)alkyl; R.sub.3 represents (C.sub.1-C.sub.6)alkyl;
R.sub.4 represents halogen or trifluoromethyl; R.sub.5 and R.sub.6
are the same or different and represent hydrogen, trifluoromethyl,
trifluoromethoxy, cyano, (C.sub.1-C.sub.6) alkyl, halogen,
(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkyl, mono or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6), or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or R.sub.4
and R.sub.5 together with the carbon atoms to which they are
attached form a 5 or 6 membered aromatic ring which is optionally
substituted with one or two groups independently selected from
halogen, nitro, trifluoromethyl, cyano, hydroxy,
(C.sub.1-C.sub.6)alkyl, amino, or mono- or
di(C.sub.1-C.sub.6)alkylamino; or OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4; and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring where up to two of the
members are optionally hetero atoms selected from oxygen, sulfur
and nitrogen, and where each member is optionally substituted with
(C.sub.1-C.sub.6)alkyl; X represents a bond or CH.sub.2, where the
CH.sub.2 is optionally mono- or disubstituted with a
(C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkoxy- ; and A, B, C.
and D are the same or different and represent CR.sub.P or N where
R.sub.p represents hydrogen or C.sub.1-C.sub.6 alkyl or
C.sub.1-C.sub.6 alkoxy where the alkyl portion of each is
optionally substituted with, carboxy, halogen, amino, or mono- or
di(C.sub.1-C.sub.6)alkylamino, with the proviso that not more than
two of A, B, C. and D represent N.
2. A compound of the formula: 24or pharmaceutically acceptable
non-toxic salts thereof wherein R.sub.1 is not 3-fluorobenzyl and
represents (i) (C.sub.2-C.sub.6)alkenyl; or (ii) R.sub.1 represents
aryl(C.sub.1-C.sub.6)alkyl or heteroaryl(C.sub.1-C.sub.6)alkyl,
where the ring portion of each is optionally substituted with one,
two or three groups independently selected from halogen, nitro,
trifluoromethyl, trifluoromethoxy, cyano, hydroxy,
(C.sub.1-C.sub.6) alkyl, hydroxy(C.sub.1-C.sub.6)alkyl, amino,
mono- or di(C.sub.1-C.sub.6)alkylam- ino, amino (C.sub.1-C.sub.6)
alkyl, mono- or di(C.sub.1-C.sub.6) alkylamino
(C.sub.1-C.sub.6)alkyl, mono- or di(C.sub.1-C.sub.6)alkylamino-
(C.sub.1-C.sub.6)alkoxy, or (iii) OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4; and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring where up to two of the
members in the ring are optionally hetero atoms selected from
oxygen, sulfur and nitrogen, and where each member is optionally
substituted with (C.sub.1-C.sub.6) alkyl; R.sub.3 is
C.sub.3-C.sub.6 alkyl; R.sub.4 is chloro or fluoro; and R.sub.a and
R.sub.b independently represent hydrogen or C.sub.1-C.sub.6
alkoxy.
3. A compound according to claim 2, wherein R.sub.1 is benzyl mono-
or disubstituted on the ring portion with (C.sub.1-C.sub.6) alkyl,
halogen, nitro, trifluoromethyl, trifluoromethoxy, cyano, hydroxy,
(C.sub.1-C.sub.6) alkyl, hydroxy(C.sub.l-C.sub.6)alkyl, amino,
mono- or di(C.sub.1-C.sub.6)alkylamino, aminomethyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkyl, or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4; and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6) alkyl; or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring where up to two of the
members are optionally hetero atoms selected from oxygen, sulfur
and nitrogen, and where each member is optionally substituted with
(C.sub.1-C.sub.6) alkyl; except that R.sub.1 is not
3-fluorobenzyl.
4. A compound according to claim 2, wherein R.sub.4 is chloro and
each of R.sub.a and R.sub.b are C.sub.1-C.sub.6 alkoxy.
5. A compound according to claim 2, wherein R.sub.3 is isoamyl and
R.sub.a and R.sub.b are methoxy.
6. A compound according to claim 2, wherein R.sub.4 is chloro,
R.sub.3 is isoamyl and R.sub.a and R.sub.b are methoxy.
7. A compound according to claim 2 wherein R.sub.1 is benzyl
substituted in the 2- or 3-positions of its phenyl ring with
hydroxy, C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.2 alkoxy,
.omega.-[4-((C.sub.1-C.sub.6)alkyl)piperaz-
inyl](C.sub.1-C.sub.4)alkoxy, methyl sulfonate, 3-halopropoxy,
carboxymethoxy, 2-, 3-, or 4-pyridylmethyl,
3-pyrrolidinyl(C.sub.1-C.sub.- 6)alkoxy, tetrazolyl, halogen,
preferably bromo, fluoro or chloro,
(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.4)alkoxy,
3-morpholin-4-yl(C.sub.1-C.sub.6)alkoxy,
.omega.-piperidyl(C.sub.1-C.sub.- 4)alkoxy,
(C.sub.1-C.sub.3)alkoxycarbonylmethoxy, [N-(methylsulfonyl)carba-
moyl]methoxy, trifluoromethyl, and nitro.
8. A compound according to claim 7, wherein R.sub.1 is a benzyl
group substituted in the 2-position of the phenyl ring.
9. A comopound according to claim 2, wherein R.sub.1 is 2-fluoro-,
2-bromo- or 2-chloro-5-nitrobenzyl, 3,5-dihalobenzyl where the
halogen is chloro or fluoro,
5-hydroxy(C.sub.1-C.sub.2)alkyl-2-(C.sub.1-C.sub.3)alko- xybenzyl,
5-(C.sub.2-C.sub.4)alkanoyl-2-(C.sub.1-C.sub.3)alkoxybenzyl, and
3-amino-5- or 6-(C.sub.1-C.sub.2)alkoxybenzyl.
10. A comopound according to claim 2, wherein R.sub.1 is alkenyl
groups such as allyl or 1-buten-2- or 3-yl.
11. A compound according to claim 1 which is: 25wherein: R.sub.1
represents aryl(C.sub.1-C.sub.6)alkyl or
heteroaryl(C.sub.1-C.sub.6)alkyl- , where the ring portion of each
is optionally substituted with one, two or three groups
independently selected from halogen, nitro, trifluoromethyl,
trifluoromethoxy, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono- or di (C.sub.1-C.sub.6)
alkylamino, aminomethyl, methylamino (C.sub.1-C.sub.6) alkyl, mono-
or di(C.sub.1-C.sub.6)alkylaminomethyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy, or OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4 and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring up to two of which members
are optionally hetero atoms selected from oxygen, sulfur and
nitrogen, and each member is optionally substituted with
(C.sub.1-C.sub.6)alkyl, with the proviso that R.sub.1 is not
3-fluorobenzyl.
12. A compound according to claim 1 which is: 26or the
pharmaceutically acceptable non-toxic salts thereof wherein:
R.sub.1 represents (C.sub.2-C.sub.6) alkenyl; or R.sub.1 represents
aryl(C.sub.1-C.sub.6)alk- yl or heteroaryl(C.sub.1-C.sub.6)alkyl,
where the ring portion of each is optionally substituted with one,
two or three groups independently selected from halogen, nitro,
trifluoromethyl, trifluoromethoxy, cyano, hydroxy,
(C.sub.1-C.sub.6)alkyl, hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono-
or di(C.sub.1-C.sub.6)alkylamino, aminomethyl,
methylamino(C.sub.1-C.sub.6)alkyl, mono- or
di(C.sub.1-C.sub.6)alkylamino- methyl, mono- or di(C.sub.1-C.sub.6)
alkylamino(C.sub.1-C.sub.6)alkoxy, or OR.sub.7,
O(CH.sub.2).sub.nC(O)R.sub.7, O(CH.sub.2).sub.nNR.sub.7R.sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where n=1, 2, 3, or 4 and
R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl or R.sub.7 and
R.sub.8 together with the nitrogen to which they are attached form
a 5, 6 or 7 membered carbocyclic ring up to two of which members
are optionally hetero atoms selected from oxygen, sulfur and
nitrogen, and each member is optionally substituted with
(C.sub.1-C.sub.6)alkyl, with the proviso that R.sub.1 is not
3-fluorobenzyl.
13. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(2-methoxyphenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbut-
yl)carboxamide.
14. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[1-({2-[3-(4-methylpiperazinyl)propoxy]phenyl}meth-
yl)benzimidazol-2-yl]methyl}carboxamide.
15. A compound according to claim 1, which is
2-{2-[(2-{[(2-chloro-3,4-dim-
ethoxyphenyl)-N-(3-methylbutyl)carbonylamino]methyl}benzimidazolyl)methyl]-
phenoxy}acetic acid.
16. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[3-(2-pyridylmethyl)imidazolo[5,4-b]pyridin-2-yl]m-
ethyl}carboxamide.
17. A compound according to claim 1, which is
2-(2-chlorophenyl)-N-({3-[(2-
-methoxyphenyl)methyl]imidazolo[5,4-b]pyridin-2-yl}methyl)-N-(3-methylbuty-
l)acetamide.
18. A compound according to claim 1, which is
[2-chloro-4-(methylethoxy)ph-
enyl]-N-({3-[(2-methoxyphenyl)methyl]imidazolo[5,4-b]pyridin-2-yl}methyl)--
N-(3-methylbutyl)carboxamide.
19. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(2-methoxyphenyl)methyl]imidazolo[4,5-c]pyridin-2-yl}methyl)-N-(-
3-methylbutyl)carboxamide.
20. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-[(1-{[3-(3-pyrrolidinylpropoxy)phenyl]methyl}benzim-
idazol-2-yl)methyl]carboxamide.
21. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-[(1-prop-2-enylbenzimidazol-2-yl)methyl]carboxamide-
.
22. A compound according to claim 1, which is
2-(2-{[(2-chloro-3,4-dimetho-
xyphenyl)-N-(3-methylbutyl)carbonylamino]methyl}-1-[(2-methoxyphenyl)methy-
l]benzimidazol-4-yloxy)acetic acid.
23. A compound according to claim 1, which is
2-{2-[(2-{[(2-chloro-3,4-dim-
ethoxyphenyl)-N-(3-methylbutyl)carbonylamino]methyl}benzimidazolyl)methyl]-
phenoxyl}-N-(methylsulfonyl) acetamide.
24. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-({1-[(2-(2H-1,2,3,4-tetraazol-5-yl)phenyl)methyl]be-
nzimidazol-2-yl}methylcarboxamide.
25. A compound according to claim 1, which is
2-(2-chlorophenyl)-N-({1-[(2-
-chlorophenyl)methyl]benzlmidazol-2-yl}methyl)-N-pentylacetamide.
26. A compound according to claim 1, which is
2-(2-chlorophenyl)-N-({1-[(2-
-chlorophenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbutyl)acetamide.
27. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny- l)-N-({1-[(2-methoxy-5-nitrophenyl)
methyl]benzimidazol-2-yl}methyl)-N-(3--
methylbutyl)carboxamide.
28. A compound according to claim 1, which is
2-chloro-3,4-dimethoxyphenyl-
)-N-({1-[(2-methoxyphenyl)methyl]benzimidazol-2-yl}methyl)-N-pentylcarboxa-
mide.
29. A compound according to claim 1, which is
N-({3-[(3,5-dichlorophenyl)m-
ethyl]imidazolo[5,4-b]pyridin-2-yl}methyl)(2-chloro-3,4-dimethoxyphenyl)-N-
-(3-methylbutyl)carboxamide.
30. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(2-hydroxyphenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbut-
yl)carboxamide.
31. A compound according to claim 1, which is
2-[(2-{[(2-chloro-3,4-dimeth-
oxyphenyl)-N-(3-methylbutyl)carbonylamino]methyl}benzimidazolyl)methyl]phe-
nyl methylsulfonate.
32. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-[(1-{[5-(hydroxyethyl)-2-methoxyphenyl]methyl}benzimidazol-2-yl)methy-
l]-N-(3-methylbutyl) carboxamide.
33. A compound according to claim 1, which is
N-({1-[(5-acetyl-2-methoxyph-
enyl)methyl]benzimidazol-2-yl}methyl)(2-chloro-3,4-dimethoxyphenyl)-N-(3-m-
ethylbutyl)carboxamide.
34. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(2-chlorophenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbuty-
l)carboxamide.
35. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-{[1-({3-[3-(methylamino)propoxy]phenyl}methyl)benzimidazol-2-yl]methy-
l}-N-(3-methylbutyl)carboxamide.
36. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[1-({3-[3-(4-methylpiperazinyl)propoxy]phenyl}meth-
yl)benzimidazol-2-yl]methyl)carboxamide.
37. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[1-(2-pyridylmethyl)benzimidazol-2-yl]methyl}carbo-
xamide.
38. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-{[1-({3-[2-(ethylamino)ethoxy]phenyl}methyl)benzimidazol-2-yl]methyl}-
-N-(3-methylbutyl) carboxamide.
39. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(2-fluorophenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbuty-
l) carboxamide.
40. A compound according to claim 1, which is
N-butyl(2-chloro-3,4-dimetho-
xyphenyl)-N-({1-[(2-methoxyphenyl)methyl]benzimidazole-2-yl}methyl)carboxa-
mide.
41. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({3-[(3-chlorophenyl)methyl]imidazolo[5,4-b]pyridin-2-yl}methyl)-N-(3-
-methylbutyl)carboxamide.
42. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-({1-[(2-methylphenyl)methyl]benzimidazol-2-yl}methy-
l)carboxamide.
43. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-[(1-{[3-(3-morpholin-4-ylpropoxy)phenyl]methyl}benz-
imidazol-2-yl)methyl]carboxamide.
44. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[1-({3-[2-(4-methylpiperazinyl)ethoxy]phenyl}methy-
l)benzimidazol-2-yl]methyl}carboxamide.
45. A compound according to claim 1, which is
3-[(2-{[(2-chloro-3,4-dimeth-
oxyphenyl)-N-(3-methylbutyl)carbonylamino]methyl}benzimidazolyl)methyl]phe-
nyl methylsulfonate.
46. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-[(1-{[3-(2-piperidylethoxy)phenyl]methyl}benzimidaz-
ol-2-yl)methyl]carboxamide.
47. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-({1-[(3-hydroxyphenyl)methyl]benzimidazol-2-yl}methyl)-N-(3-methylbut-
yl)carboxamide.
48. A compound according to claim 1, which is ethyl
2-{2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-carbonylamino-
]methyl}benzimidazolyl)methyl]phenoxy}acetate.
49. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny- l)-N-({3-[2-methoxyphenyl)
methyl]imidazolo [5,4-b]pyridin-2-yl}methyl)-N--
(3-methylbutyl)carboxamide.
50. A compound according to claim 1, which is
N-({1-[(3-amino-6-methoxyphe-
nyl)methyl]benzimidazol-2-yl}methyl)(2-chloro-3,4-dimethoxyphenyl)-N-(3-me-
thylbutyl) carboxamide.
51. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-{[1-({2-[2-(4-methylpiperazinyl)ethoxy]phenyl}methy-
l)benzimidazol-2-yl]methyl}carboxamide.
52. A compound according to claim 1, which is
N-butyl(2-chloro-3,4-dimetho-
xyphenyl)-N-({1-[(3-fluorophenyl)methyl]benzimidazol-2-yl}methyl)carboxami-
de.
53. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-(3-methylbutyl)-N-[(1-{[2-(trifluoromethyl)phenyl]methyl}benzimidazol-
-2-yl)methyl]carboxamide.
54. A compound according to claim 1, which is
[2-chloro-4-(methylethoxy)ph-
enyl]-N-(3-methylbutyl)-N-({1-[(2-nitrophenyl)methyl]benzimidazol-2-yl}met-
hylcarboxamide.
55. A compound according to claim 1, which is
(2-chloro-3,4-dimethoxypheny-
l)-N-[(4-methyoxy-1-prop-2-enylbenzimidazol-2-yl)methyl]-N-(3-methylbutyl)-
carboxamide.
56. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable carrier or excipient.
57. A method of treatment or diagnosis of a patient suffering from
physiological disorders associated with an excess amount of
bradykinin comprising administering to the patient a suffienct
amount of a compound according to claim 1 to reduce the effects of
excess bradykinin.
58. A method of treatment or diagnosis of a patient suffering from
physiological disorders associated with an insufficient amount of
bradykinin comprising administering to the patient a sufficient
amount of a compound according to claim 1 to reduce the effects of
insufficient bradykinin.
59. A method according to claim 57 wherein the physiological
disorders are renal diseases, heart failure, hypertension,
Meniere's disease, vaginal inflammation, peripheral circulatory
disorders, climacteric disturbance, retinochoroidal circulatory
disorders, myocardial ischemia, myocardial infarction,
postmyocardial infarction syndrome, angina pectoris, restenosis
after percutaneous transluminal coronary angioplasty, hepatitis,
liver cirrhosis, pancreatitis, ileus, diabetes, diabetic
complications, male infertility, glaucoma, increase of permeability
of blood-brain barrier, pain, asthma or rhinitis.
60. A method according to claim 58 wherein the physiological
disorders are renal diseases, heart failure, hypertension,
Meniere's disease, vaginal inflammation, peripheral circulatory
disorders, climacteric disturbance, retinochoroidal circulatory
disorders, myocardial ischemia, myocardial infarction,
postmyocardial infarction syndrome, angina pectoris, restenosis
after percutaneous transluminal coronary angioplasty, hepatitis,
liver cirrhosis, pancreatitis, ileus, diabetes, diabetic
complications, male infertility, glaucoma, increase of permeability
of blood-brain barrier, pain, asthma or rhinitis.
61. A use of a compound according to claim 1 in the manufacture of
a medicament for the treatment of renal diseases, heart failure,
hypertension, Meniere's disease, vaginal inflammation, peripheral
circulatory disorders, climacteric disturbance, retinochoroidal
circulatory disorders, myocardial ischemia, myocardial infarction,
postmyocardial infarction syndrome, angina pectoris, restenosis
after percutaneous transluminal coronary angioplasty, hepatitis,
liver cirrhosis, pancreatitis, ileus, diabetes, diabetic
complications, male infertility or glaucoma, increase of
permeability of blood-brain barrier, pain, asthma or rhinitis.
62. A process for preparing a compound according to claim 1.
63. A method for the treatment or prevention of a disease or
disorder associated with pathogenic BK-2 receptor activation, said
method comprising administering to a patient in need of such
treatment or prevention an effective amount of a compound of claim
1.
64. The use of a compound according to claim 1 for the manufacture
of a medicament for the treatment or prevention of a disease or
disorder associated with pathogenic bradykinin-2 receptor
activation.
65. The use of a compound according to claim 1 for the manufacture
of a medicament for the treatment or prevention of pain, asthma, or
rhinitis.
66. A method according to claim 63 wherein the disorder associated
with pathogenic bradykinin receptor activation is pain, asthma or
rhinitis
67. A method for localizing BK-2 receptors in a tissue sample
comprising: contacting the sample with a detectably-labeled
compound of claim 1 under conditions that permit binding of the
compound to neurokinin 3 receptors, washing the sample to remove
unbound compound, and detecting the bound compound.
68. A method of inhibiting the binding of a bradykinin to a BK-2
receptor, said method comprising contacting a compound of claim 1
with cells expressing the BK-2 receptor in the presence of a
bradykinin, wherein the compound is present at a concentration
sufficient to inhibit bradykinin binding to cells expressing a
cloned human BK-2 receptor in vitro.
69. A method for altering the signal-transducing activity of BK-2
receptors, said method comprising exposing cells expressing BK-2
receptors to a compound according to claim 1 at a concentration
sufficient to inhibit bradykinin binding to cells expressing a
cloned human BK-2 receptor in vitro.
70. A packaged pharmaceutical composition comprising the
pharmaceutical composition of claim 56 in a container and
instructions for using the composition to treat a patient suffering
from a disorder responsive to BK-2 receptor modulation.
71. The packaged pharmaceutical composition of claim 70, wherein
said patient is suffering from anxiety, depression, schizoprenia,
obesity, chronic pulmonary obstructive disorder, or pain.
72. A compound according to claim 1 wherein the compound exhibits
an IC.sub.50 of 1 micromolar or less in a standard assay of BK-2
receptor binding.
73. A compound according to claim 1 wherein the compound exhibits
an IC.sub.50 of 100 nanomolar or less in a standard assay of BK-2
receptor binding.
74. A compound according to claim 1 wherein the compound exhibits
an IC.sub.50 of 10 nanomolar or less in a standard assay of BK-2
receptor binding.
75. A method of increasing the permeability of the blood brain
barrier comprising administering a compound according to claim 1 to
a patient.
76. A method of increasing the brain concentration of a CNS active
compound comprising administering a compound according to claim 1
and the CNS active compound to a patient.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/127,505, filed Apr. 2, 1999.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to aryl and heteroaryl fused
aminoalkylimidazole derivatives which, when appropriately
substituted, are selective modulators of Bradykinin B.sub.2
receptors. This invention also relates to pharmaceutical
compositions comprising such compounds. It further relates to the
use of such compounds in treating a variety of central and
peripheral disorders. Additionally, compounds of this invention are
useful as positive controls in assays for BK-2 receptor activity
and when appropriately labeled as probes for the localization of
BK-2 receptors in tissue sections.
[0004] 2. Background
[0005] Bradykinin (BK), a nonapeptide, and the closely related
decapeptide kallidin (Lys-BK), are produced by proteolytic cleavage
of high molecular weight kininogen by plasma kallikreins (Bhoola et
al., Pharmacol. Rev. 1992, 1-80; Regoli et al. Pharmacol. Rev. 1980
1-46; Bathon & Proud, Ann. Rev. Pharmac. Toxic. 1991, 129-162).
The effects of bradykinin and kallidin are mediated by specific
seven transmembrane G-protein coupled receptors.
[0006] The existence of two bradykinin receptor subtypes was
initially proposed by Regoli and Barabe (Pharmacol. Rev., 1980,
1-46) and this hypothesis had been unequivocally confirmed within
the last six years. The expression and cloning of a rat bradykinin
receptor, now known to be a BK-2 receptor, was first reported by
McEachern et al. (PNAS 1991, 88(17):7724-7728). Hess, et al.
(Biochem Biophys. Res. Commun. 1992, 260-268) reported the cloning
and pharmacological characterization of a human BK-2 receptor.
Menke, et al. (J. Biol. Chem. 1994, 21583-21586) describes the
expression and cloning of a human bradykinin (B.sub.1)
receptor.
[0007] Both BK and kallidin activate the B.sub.2 receptor while
only kallidin is active at the B.sub.1 receptor. However, both
compounds are rapidly cleaved to produce B.sub.1 receptor agonists,
and then further degraded by kinases to produce inactive peptides.
The instability of BK and kallidin suggests that these peptides act
locally. Both receptors are expressed in a number of peripheral
tissues as well as in the CNS.
[0008] The B.sub.2 receptor is expressed constitutively in a
variety of tissues (Regoli et al., Eur. J. Pharmacol., 1981,
105-115) and accounts for the majority of the acute pharmacological
effects of bradykinin. The B.sub.1 receptor is inducibly expressed
(Regoli et al., Eur. J. Pharmacol., 1981, 105-115; Deblois et al.,
Immunopharmacology, 1989, 187-98; Marceau, Immunopharmacology,
1995, 1-26.) and appears to act predominantly in pathophysiological
conditions (Dray and Perkins, J. Neurophysiol., 1993, 256-272). The
BK-1 receptor has been especially implicated in persistent
hyperalgesia and chronic inflammation.
[0009] Bradykinin is an effector of a number of inflammatory
responses including bronchoconstriction, plasma extravasation,
release of prostaglandins/leukotrienes, smooth muscle
contraction/relaxation and nociception (Burch et al., Med. Res.
Rev. 1990, 237-269). Bradykinin and the related peptide kallidin
have been implicated in a number of disease conditions, including
but not limited to pain (Whalley et al., Naunyn. Schmiedeberg's
Arch. Pharmc., 1987, 652-655), rhinitis, anaphylaxis, inflammatory
bowel disease, vascular permeability (Schachter et al., Br. J.
Pharmc., 1987, 851-855; Whalley et al., Naunyn Schmiedeberg's Arch.
Pharmc., 1987, 430-433), algesia, vasodilataion, inflammatory
response (Burch & De Haas, Naunyn Schmiedeberg's Arch. Pharmc.
1990, 189-193), hypotension associated with sepsis (Sharma et al.,
Agents Actions, 1992, 258-269), bronchopulmonary disorders
including asthma (Jin et al., Br. J. Pharmac., 1989, 598-602), and
increased cell proliferation. Antagonists of the BK-2 receptor are
useful in treating these conditions. Additionally bradykinin has
been implicated in increased glucose uptake, and decreased blood
glucose concentration (Henriksen et al., Diabetes, 1996, S125-S128;
Yang et al., J Pharmacol. Exp. Ther., 1997, 1247-1252). Therefore
agonists of the BK-2 receptor may be useful in the treatment of
Type II diabetes. Unterberg et al. (J Cereb. Blood Flow Metab.,
1984, 574-585) report an increased permeability of the blood-brain
barrier due to bradykinin. Thus, agonists of the BK-2 receptor
could also be used to increase the brain levels of pharmaceutical
compounds used to treat central nervous system disorders when
administered with these compounds. Therefore, compounds that
modulate the bradykinin B.sub.2 (BK-2) receptor as agonists or
antagonists would have considerable therapeutic benefit.
[0010] A number of tissues and cultured cell lines has been
assessed for the presence of bradykinin receptors using
radiolabeled bradykinin or a radiolabeled bradykinin analogue as a
probe (See Hall, Gen. Pharma., 1997, 28: 1-6, for a compilation of
such studies.). Although bradykinin and its analogues exhibit high
affinity for bradykinin receptors there are some difficulties in
using these ligands as receptor localization probes. Bradykinin
binds to both BK-1 and BK-2 receptors and therefore cannot be used
to distinguish receptor subtypes. Also bradykinin and many of its
peptide analogues are susceptible to rapid degradation by
kininases, leading to experimental difficulties. Nonpeptidic
ligands are not susceptible to kininase activity. Therefore, small
molecules that bind with high affinity and high selectivity to BK-2
receptors are especially desirable tools for BK-2 localization
studies.
SUMMARY OF THE INVENTION
[0011] This invention provides compounds of Formula I (shown below)
and pharmaceutical compositions comprising compounds of Formula I.
Preferred compounds of the invention exhibit high selectivity for
G-coupled protein receptors, especially bradykinin B.sub.2
receptors. Preferred compounds of Formula I also bind with high
affinity to these receptors.
[0012] The invention further provides methods of treating patients
suffering from certain inflammatory disorders and other conditions
mediated by bradykinin. The invention also provides methods of
treating patients (humans and non-humans) suffering from conditions
in which agonism of the BK-2 receptor may prove beneficial.
Treatment of humans, domesticated companion animals (pets) or
livestock animals suffering such conditions with an effective
amount of a compound of the invention is contemplated by the
invention.
[0013] In a separate aspect, the invention provides methods of
using compounds of this invention as positive controls in assays
for BK-2 receptor activity and using appropriately labeled
compounds of the invention as probes for the localization of BK-2
receptors in tissue sections.
[0014] Accordingly, in one aspect, the invention is directed to
compounds of Formula I: 2
[0015] wherein:
[0016] R.sub.1 is not 3-fluorobenzyl and represents
[0017] (i) (C.sub.2-C.sub.6) alkenyl; or
[0018] (ii) R.sub.1 represents aryl (C.sub.1-C.sub.6) alkyl or
heteroaryl (C.sub.1-C.sub.6) alkyl, where the ring portion of each
is optionally substituted with one, two or three groups
independently selected from halogen, nitro, trifluoromethyl,
trifluoromethoxy, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono- or
di(C.sub.1-C.sub.6)alkylamino, amino(C.sub.1-C.sub.6)alkyl, mono-
or di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy, or
[0019] (iii) OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.sub.8, O(CH.sub.2).sub.nCO.sub.2R.sub.7,
NR.sub.7COR.sub.8, COR.sub.7, CONR.sub.7R.sub.8 or CO.sub.2R.sub.7
where
[0020] n=1, 2, 3, or 4 and
[0021] R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or
[0022] R.sub.7 and R.sub.8 together with the nitrogen to which they
are attached form a 5, 6 or 7 membered carbocyclic ring where up to
two of the members in the ring are optionally hetero atoms selected
from oxygen, sulfur and nitrogen, and where each member is
optionally substituted with (C.sub.1-C.sub.6) alkyl;
[0023] R.sub.2 represents hydrogen, hydroxy, halogen,
trifluoromethyl, trifluoromethoxy, amino(C.sub.1-C.sub.6)alkyl,
mono- or di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6), mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6) alkoxy; or
[0024] OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.- sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where
[0025] n-1, 2, 3, or 4; and
[0026] R.sub.7 and R.sub.8 are the same or different and represent
is hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or
[0027] R.sub.7 and R.sub.8 together with the nitrogen to which they
are attached form a 5, 6 or 7 membered carbocyclic ring where up to
two of the members are optionally hetero atoms selected from
oxygen, sulfur and nitrogen, and where each member is optionally
substituted with (C.sub.1-C.sub.6) alkyl;
[0028] R.sub.3 represents (C.sub.1-C.sub.6)alkyl;
[0029] R.sub.4 represents halogen or trifluoromethyl;
[0030] R.sub.5 and R.sub.6 are the same or different and represent
hydrogen, trifluoromethyl, trifluoromethoxy, cyano,
(C.sub.1-C.sub.6)alkyl, halogen,
(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.su- b.6)alkyl, mono or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6), or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or
[0031] R.sub.4 and R.sub.5 together with the carbon atoms to which
they are attached form a 5 or 6 membered aromatic ring which is
optionally substituted with one or two groups independently
selected from halogen, nitro, trifluoromethyl, cyano, hydroxy,
(C.sub.1-C.sub.6)alkyl, amino, or mono- or
di(C.sub.1-C.sub.6)alkylamino; or
[0032] OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.- sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where
[0033] n=1, 2, 3, or 4; and
[0034] R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or
[0035] R.sub.7 and R.sub.8 together with the nitrogen to which they
are attached form a 5, 6 or 7 membered carbocyclic ring where up to
two of the members are optionally hetero atoms selected from
oxygen, sulfur and nitrogen, and where each member is optionally
substituted with (C.sub.1-C.sub.6) alkyl;
[0036] X represents a bond or CH.sub.2, where the CH.sub.2 is
optionally mono- or disubstituted with a (C.sub.1-C.sub.6)alkyl or
(C.sub.1-C.sub.6)alkoxy; and
[0037] A, B, C. and D are the same or different and represent CH or
N with the proviso that not more than two of A, B, C. and D
represent N.
[0038] Preferred compounds of the inventions are modulators of
G-coupled protein receptors, especially BK-2 receptors. These
compounds are therefore useful in the diagnosis and treatment of
renal diseases, heart failure, hypertension, Meniere's disease,
vaginal inflammation and pain, peripheral circulatory disorders,
climacteric disturbance, retinochoroidal circulatroy disorders,
myocardial ischemia, myocardial infarction, postmyocardial
infarction syndrome, angina pectoris, restenosis after percutaneous
transluminal coronary angioplasty, hepatitis, liver cirrhosis,
pancreatitis, ileus, diabetes, diabetic complications, male
infertility or glaucoma, or for the increase of permeability of
blood-brain barrier, pain, asthma and rhinitis.
[0039] In another aspect, the invention provides methods for
treating and/or preventing the above-listed disorders, which
methods comprise administration to a patient in need thereof of an
effective amount of a compound of Formula I.
[0040] In yet another aspect, the invention provides intermediates
useful in the preparation of the compounds of Formula I.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The compounds encompassed by the instant invention are
represented by general Formula I set forth above and include the
pharmaceutically acceptable non-toxic salts thereof.
[0042] In addition, the present invention also encompasses
compounds of Formula II 3
[0043] wherein R.sub.1 is as defined above for Formula I; and
[0044] R.sub.3 is C.sub.3-C.sub.6 alkyl, preferably n-butyl,
isoamyl, or n-pentyl;
[0045] R.sub.4 is chloro or fluoro; and
[0046] R.sub.a and R.sub.b independently represent hydrogen or
C.sub.1-C.sub.6 alkoxy.
[0047] More preferred compounds of Formula II are where R.sub.1 is
benzyl mono- or disubstituted on the ring portion with
[0048] (C.sub.1-C.sub.6)alkyl, halogen, nitro, trifluoromethyl,
trifluoromethoxy, cyano, hydroxy, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono- or
di(C.sub.1-C.sub.6)alkylam- ino, aminomethyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)a- lkyl, or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or
[0049] OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.- sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where
[0050] n-1, 2, 3, or 4; and
[0051] R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SO.sub.2Me, or (C.sub.1-C.sub.6) alkyl; or
[0052] R.sub.7 and R.sub.8 together with the nitrogen to which they
are attached form a 5, 6 or 7 membered carbocyclic ring where up to
two of the members are optionally hetero atoms selected from
oxygen, sulfur and nitrogen, and where each member is optionally
substituted with (C.sub.1-C.sub.6) alkyl;
[0053] except that R.sub.1 is not 3-fluorobenzyl.
[0054] Even more preferred compounds of Formula II are those where
R.sub.4 is chloro and R.sub.a and R.sub.b are independently
C.sub.1-C.sub.6 alkoxy, most preferably C.sub.1-C.sub.3 alkoxy.
Particularly preferred compounds of Formula II are those where
R.sub.3 is butyl or isoamyl, i.e, 3-methylbutyl, R.sub.4 is chloro,
and R.sub.a and R.sub.b are independently C.sub.1-C.sub.2 alkoxy,
most preferably methoxy.
[0055] In addition, the present invention encompasses compounds of
the Formula III. 4
[0056] wherein R.sub.1 is as defined above for Formula I; and
[0057] R.sub.3 is C.sub.3 or C.sub.6 alkyl, preferably n-butyl,
isoamyl, or n-pentyl;
[0058] R.sub.4 is chloro or fluoro; and
[0059] R.sub.a and R.sub.b independently represent hydrogen or
C.sub.1-C.sub.6 alkoxy.
[0060] More preferred compounds of Formula II are where R.sub.1 is
benzyl mono- or disubstituted on the ring portion with
[0061] (C.sub.1-C.sub.6)alkyl, halogen, nitro, trifluoromethyl,
trifluoromethoxy, cyano, hydroxy, (C.sub.1-C.sub.6) alkyl,
hydroxy(C.sub.1-C.sub.6)alkyl, amino, mono- or
di(C.sub.1-C.sub.6)alkylam- ino, aminomethyl, mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)a- lkyl, or mono- or
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkoxy; or
[0062] OR.sub.7, O(CH.sub.2).sub.nC(O)R.sub.7,
O(CH.sub.2).sub.nNR.sub.7R.- sub.8,
O(CH.sub.2).sub.nCO.sub.2R.sub.7, NR.sub.7COR.sub.8, COR.sub.7,
CONR.sub.7R.sub.8 or CO.sub.2R.sub.7 where
[0063] n-1, 2, 3, or 4; and
[0064] R.sub.7 and R.sub.8 are the same or different and represent
hydrogen, SC.sub.2Me, or (C.sub.1-C.sub.6)alkyl; or
[0065] R.sub.7 and R.sub.8 together with the nitrogen to which they
are attached form a 5, 6 or 7 membered carbocyclic ring where up to
two of the members are optionally hetero atoms selected from
oxygen, sulfur and nitrogen, and where each member is optionally
substituted with (C.sub.1-C.sub.6) alkyl;
[0066] except that R.sub.1 is not 3-fluorobenzyl.
[0067] Even more preferred compounds of Formula III are those where
R.sub.4 is chloro and R.sub.a and R.sub.b are independently
C.sub.1-C.sub.6 alkoxy, most preferably C.sub.1-C.sub.3 alkoxy.
Particularly preferred compounds of Formula III are those where
R.sub.3 is butyl or isoamyl, i.e, 3-methylbutyl, R.sub.4 is chloro,
and R.sub.a and R.sub.b are methoxy.
[0068] Particularly preferred R, groups in Formulae II and III are
benzyl substituted in the 2- or 3-positions of its phenyl ring with
hydroxy, C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.2 alkoxy,
.omega.-[4-((C.sub.1-C.sub-
.6)alkyl)piperazinyl](C.sub.1-C.sub.4)alkoxy, methyl sulfonate,
3-halopropoxy, carboxymethoxy, 2-, 3-, or
4-pyridyl(C.sub.1-C.sub.6)alkyl- , preferably 2-, 3-, or
4-pyridyl(C.sub.1-C.sub.2)alkyl,
3-pyrrolidinyl(C.sub.1-C.sub.6)alkoxy; tetrazolyl, halogen,
preferably bromo, fluoro or chloro, alkylamino (C.sub.1-C.sub.6)
alkoxy, preferably 3-(methylamino)propoxy or 2-(ethylamino)ethoxy,
morpholinyl(C.sub.1-C.sub- .6) alkoxy, preferably
3-morpholin-4-ylpropoxy or 2-(morpholin-4-yl)ethoxy- ,
.omega.-piperidyl(C.sub.1-C.sub.4)alkoxy,
(C.sub.1-C.sub.3)alkoxycarbony- lmethoxy, trifluoromethyl,
(N-(methylsulfonyl) carbamoyl)methoxy, and nitro.
[0069] The most preferred among these 2- or 3-substituted benzyl
groups are those substituted in the 2-position of the phenyl
ring.
[0070] Other particularly preferred R, groups of the invention are
2-fluoro-, 2-bromo- or 2-chloro-5-nitrobenzyl, 3,5-dihalobenzyl
where the halogen is chloro or fluoro,
5-hydroxy(C.sub.1-C.sub.2)alkyl-2-(C.sub.1-C- .sub.3)alkoxybenzyl,
5-(C.sub.2-C.sub.4)alkanoyl-2-(C.sub.1-C.sub.3)alkoxy- benzyl, and
3-amino-5-or 6-(C.sub.1-C.sub.2)alkoxybenzyl.
[0071] Still other preferred R.sub.1 groups include alkenyl groups
such as allyl or 1-buten-2-or 3-yl.
[0072] Other particularly preferred R.sub.1 groups include 2-or
3-pyridyl.
[0073] By .omega.-substitution as used herein is meant the terminal
position on, for example, an alkyl chain. Examples of such groups
are 3-hydroxypropyl, 5-morpholin-4-ylpentyl, 3-piperazinylpropoxy,
and 4-methoxybutyl.
[0074] By "alkyl", "lower alkyl", and "(C.sub.1-C.sub.6)alkyl" in
the present invention is meant straight or branched chain alkyl
groups having 1-6 carbon atoms, such as, methyl, ethyl, propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,
isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and
3-methylpentyl.
[0075] By "alkoxy", "lower alkoxy", and "(C.sub.1-C.sub.6)alkoxy"
in the present invention is meant straight or branched chain alkoxy
groups having 1-6 carbon atoms, such as, for example, methoxy,
ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,
pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy,
3-hexoxy, and 3-methylpentoxy.
[0076] By the term "halogen" in the present invention is meant
fluorine, bromine, chlorine, and iodine.
[0077] By the term "patient" is meant human patients as well as
domestic companion animals (pets) and livestock animals.
[0078] By "heteroaryl" is meant one or more aromatic ring systems
of 5-, 6-, or 7-membered rings containing at least one and up to
four heteroatoms selected from nitrogen, oxygen, or sulfur. Such
heteroaryl groups include, for example, thienyl, furanyl,
thiazolyl, imidazolyl, (is)oxazolyl, pyridyl, pyrimidinyl,
(iso)quinolinyl, napthyridinyl, benzimidazolyl, benzoxazolyl.
Preferred heteroaryls are thiazolyl and pyridyl.
[0079] By "aryl" is meant an aromatic carbocyclic group having a
single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or
multiple condensed rings in which at least one is aromatic, (e.g.,
1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl),
which is optionally mono-, di-, or trisubstituted with, e.g.,
halogen, lower alkyl, lower alkoxy, lower alkylthio,
trifluoromethyl, lower acyloxy, aryl, heteroaryl, and hydroxy. A
preferred aryl is phenyl.
[0080] Preferred (C.sub.1-C.sub.6)alkylamino groups are methylamino
and ethylamino; preferred di(C.sub.1-C.sub.6)alkylamino groups are
diethylamino and dimethylamino; preferred
amino(C.sub.1-C.sub.6)alkyl groups are aminomethyl and
2-aminoethyl; preferred mono- and
di(C.sub.1-C.sub.6)alkylamino(C.sub.1-C.sub.6)alkyl groups are
methylaminomethyl, dimethylaminomethyl, ethylaminomethyl; and
2-(ethylamino)ethyl.
[0081] Representative compounds of the invention are shown below in
Table 1.
1TABLE 1 5 6 7 8 9 10 11 12
[0082] In certain situations, the compounds of Formula I may
contain one or more asymmetric carbon atoms, so that the compounds
can exist in different stereoisomeric forms. These compounds can
be, for example, racemates or optically active forms. In these
situations, the single enantiomers, i.e., optically active forms,
can be obtained by asymmetric synthesis or by resolution of the
racemates. Resolution of the racemates can be accomplished, for
example, by conventional methods such as crystallization in the
presence of a resolving agent, or chromatography, using, for
example a chiral HPLC column.
[0083] Representative compounds of the present invention, which are
encompassed by Formula I, include, but are not limited to the
compounds described in the Examples and their pharmaceutically
acceptable acid addition salts. In addition, if the compound of the
invention is obtained as an acid addition salt, the free base can
be obtained by basifying a solution of the acid salt. Conversely,
if the product is a free base, an addition salt, particularly a
pharmaceutically acceptable addition salt, may be produced by
dissolving the free base in a suitable organic solvent and treating
the solution with an acid, in accordance with conventional
procedures for preparing acid addition salts from base
compounds.
[0084] Non-toxic pharmaceutical salts include salts of acids such
as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic,
formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric,
tartaric, maleic, hydroiodic, alkanoic such as acetic,
HOOC--(CH.sub.2)n-COOH where n is 0-4, and the like. Those skilled
in the art will recognize a wide variety of non-toxic
pharmaceutically acceptable addition salts.
[0085] The present invention also encompasses the acylated prodrugs
of the compounds of Formula I. Those skilled in the art will
recognize various synthetic methodologies which may be employed to
prepare non-toxic pharmaceutically acceptable addition salts and
acylated prodrugs of the compounds encompassed by Formula I.
[0086] Selective agonists or antagonists of the bradykinin B.sub.2
receptor provide compounds useful in treatment of renal diseases,
heart failure, hypertension, Meniere's disease, vaginal
inflammation and pain, peripheral circulatory disorders,
climacteric disturbance, retinochoroidal circulatroy disorders,
myocardial ischemia, myocardial infarction, postmyocardial
infarction syndrome, angina pectoris, restenosis after percutaneous
transluminal coronary angioplasty, hepatitis, liver cirrhosis,
pancreatitis, ileus, diabetes, diabetic complications, male
infertility or glaucoma, or for the increase of permeability of
blood-brain barrier, pain, asthma, rhinitis. The invention provides
methods of treating patients suffering from such disorders with an
amount of a compound of the invention sufficient to reduce the
symptoms of the disorder.
[0087] Bradykinin has been shown to increase the permeability of
blood-brain barrier and blood-brain tumor barrier. The invention
provides a method of increasing the brain concentration of a CNS
active compound which comprises administering to a patient in need
of such treatment a compound of the invention, that is a selective
agonist of the BK-2 receptor, along with a CNS active compound, and
thereby increasing the brain concentration of the CNS active
compound. In a particularly preferred embodiment the invention
provides a method of increasing the brain concentration of
anti-cancer and anti-tumor agents which comprises administering a
patient suffering from brain cancer or a brain tumor a compound of
the invention that is a selective agonist of the BK-2 receptor,
along with an anti-cancer and anti-tumor agent, and thereby
increasing the brain concentration of the anti-cancer or anti-tumor
agent.
[0088] The compounds of general Formula I may be administered
orally, topically, parenterally, by inhalation or spray or rectally
in dosage unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques. In addition, there is provided a pharmaceutical
formulation comprising a compound of general Formula I and a
pharmaceutically acceptable carrier. One or more compounds of
general Formula I may be present in association with one or more
non-toxic pharmaceutically acceptable carriers and/or diluents
and/or adjuvants and if desired other active ingredients. The
pharmaceutical compositions containing compounds of general Formula
I may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsion, hard or soft capsules, or syrups or
elixirs.
[0089] Compositions intended for oral use may be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient in admixture
with non-toxic pharmaceutically acceptable excipients which are
suitable for the manufacture of tablets. These excipients may be
for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate may be
employed.
[0090] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0091] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents may be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0092] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide palatable oral preparations. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0093] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0094] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monoleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monoleate. The emulsions may also contain sweetening and flavoring
agents.
[0095] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleaginous
suspension. This suspension may be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono-or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0096] The compounds of general Formula I may also be administered
in the form of suppositories for rectal administration of the drug.
These compositions can be prepared by mixing the drug with a
suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
are cocoa butter and polyethylene glycols.
[0097] Compounds of general Formula I may be administered
parenterally in a sterile medium. The drug, depending on the
vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anesthetics, preservatives and buffering agents can be dissolved in
the vehicle.
[0098] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
patient per day). The amount of active ingredient that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. Dosage unit forms will generally contain between
from about 1 mg to about 500 mg of an active ingredient.
[0099] Frequency of dosage may also vary depending on the compound
used and the particular disease treated. However, for treatment of
most disorders, a dosage regimen of 4 times daily or less is
preferred. For the treatment of chronic conditions, a dosage
regimen of 1 or 2 times daily is particularly preferred. For the
treatment of acute disorders, a single dose that rapidly reaches
effective concentrations is desirable.
[0100] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the particular disease undergoing therapy.
[0101] Preferred compounds of the invention will have certain
pharmacological properties. Such properties include, but are not
limited to oral bioavailability, low toxicity, low serum protein
binding and desirable in vitro and in vivo half-lifes. Penetration
of the blood brain barrier for compounds used to treat CNS
disorders is necessary, while low brain levels of compounds used to
treat periphereal disorders are often preferred.
[0102] Assays may be used to predict these desirable
pharmacological properties. Assays used to predict bioavailability
include transport across human intestinal cell monolayers,
including Caco-2 cell monolayers. Toxicity to cultured hepatocyctes
may be used to predict compound toxicity. Penetration of the blood
brain barrier of a compound in humans may be predicted from the
brain levels of the compound in laboratory animals given the
compound intravenously.
[0103] Serum protein binding may be predicted from albumin binding
assays. Such assays are described in a review by Oravcov, et al.
(Journal of Chromatography B (1996) volume 677, pages 1-27).
[0104] Compound half-life is inversely proportional to the
frequency of dosage of a compound. In vitro half-lifes of compounds
may be predicted from assays of microsomal half-life as described
by Kuhnz and Gieschen (Drug Metabolism and Disposition, (1998)
volume 26, pages 1120-1127).
[0105] The present invention also pertains to packaged
pharmaceutical compositions for treating disorders responsive to
BK-2 receptor modulation, e.g., treatment asthma, pain or rhinitis
by BK-2 receptor modulation. The packaged pharmaceutical
compositions include a container holding a therapeutically
effective amount of at least one BK-2 receptor modulator as
described supra and instructions for using the treating disorder
responsive to BK-2 receptor modulation in the patient.
[0106] The present invention also pertains to methods of inhibiting
the binding of bradykinin to the BK-2 receptor which methods
involve contacting a compound of the invention with cells
expressing BK-2 receptors, wherein the compound is present at a
concentration sufficient to inhibit bradykinin binding to cells
expressing a cloned human Bradykinin receptor in vitro and to
methods for altering the signal-tranducing activity of BK-2
receptors, said method comprising exposing cells expressing such
receptor to an effective amount of a compound of the invention.
[0107] The invention furthermore provides methods of using
compounds of this invention as positive controls in assays for
receptor activity and using appropriately labeled compounds of the
invention as probes for the localization of receptors, particularly
BK-2 receptors, in tissue sections. Such probes are useful for in
vitro studies, such as binding assays and autoradiography of tissue
sections and for in vivo techniques such as PET and SPECT
scans.
[0108] Compounds of the invention can be prepared using the
reactions depicted in Schemes I to VII. In Schemes I-VII, the
groups R.sub.1, R.sub.3, R.sub.7, R.sub.8 and X are as defined in
general Formula I. The numbers appearing below or adjacent the
chemical structures in these schemes refer to intermediates and are
not to be confused with the compound numbers found in the examples.
13 14 15 16 17 18 19
[0109] Those having skill in the art will recognize that the
starting materials may be varied and additional steps employed to
produce compounds encompassed by the present invention, as
demonstrated by the following examples.
[0110] The disclosures of all articles and references mentioned in
this application, including patents, are incorporated herein by
reference.
[0111] The invention is illustrated further by the following
examples which are not to be construed as limiting the invention in
scope or spirit to the specific procedures described in them.
[0112] The starting materials and various intermediates may be
obtained from commercial sources, prepared from commercially
available organic compounds, or prepared using well known synthetic
methods.
[0113] Representative examples of methods for preparing
intermediates of the invention are set forth below.
EXAMPLE 1
General Procedure for the Preparation of chloromethylbenzimidazoles
as Outlined in Scheme I
[0114] 1. Imidate hydrochloride:
[0115] A solution of 150 mL (2.37 mole) of chloroacetonitrile, 139
mL (2.37 mole) of ethanol in 1,200 mL of dry benzene is cooled to
0.degree. C. in an ice/ethanol bath. Dry HCl gas is bubbled through
the vigorously stirred solution for approximately 30 min. while the
internal temperature is maintained below 10.degree. C. The solution
is allowed to stand at room temperature overnight. The resulting
solid is filtered and washed with 2L of dry ether and allowed to
air dry to afford 328 g (88%) of imidate hydrochloride.
[0116] 2.
1-{[2-(chloromethyl)benzimidazolyl]methyl}-2-methoxybenzene: 20
[0117] A solution of 31 g (0.14 mole) of (2-aminophenyl)
[(2-methoxyphenyl)methyl]amine in 200 mL of anhydrous CHCl.sub.3 is
treated with 44 g (0.28 mole) of imidate at room temperature. The
heterogeneous reaction mixture is allowed to stir for 1 hour at
room temperature at which time no starting material is detectable
by TLC. 100 mL of saturated NaHCO.sub.3 is added and extracted
3.times.100 mL of CH.sub.2Cl.sub.2. The extracts are dried over
anhydrous Na.sub.2SO.sub.4, the solvent removed in vacuo, and the
residue chromatgraphed (SiO.sub.2) with 50% ethyl acetate/hexane to
afford 20 g (50%) of
1-{[2-(chloromethyl)benzimidazolyl]-methyl}-2-methoxybenzene. Mass
Spec M.sup.+ 287.
EXAMPLE 2
General Procedure for the Preparation of benzimidazoles as Shown in
Scheme II
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxyphenyl)methyl]benzimidazol-
-2-yl}methyl)-N-(3-methylbutyl)carboxamide
[0118] 21
[0119] A solution of 5.4 mmole
1-{[2-(chloromethyl)-benzimidazolyl]methyl}- -2-methoxybenzene in
20 mL of dry acetonitrile is treated with 10 mL of isoamylamine for
16 hours at room temperature. The solvent is removed in vacuo and
the residue is partitioned between 30 mL of ethyl acetate and 10 mL
of 1 N NaOH. The ethyl acetate layer is dried over anhydrous
Na.sub.2SO.sub.4 and solvent removed in vacuo to afford 1.7 g 97%
({1-[(2-methoxyphenyl)methyl]benzimidazol-2-yl}methyl)(3-methylbutyl)
amine. 2-Chloro-3,4-dimethoxybenzoylchloride (1.5 equ.) is treated
with 1.0 equivalent of
({1-[(2-methoxyphenyl)methyl]benzimidazol-2-yl}methyl)(-
3-methylbutyl) amine in dichloromethane at room temperature for 1
hour. The reaction is quenched with 1 N NaOH and partitioned
between dichloromethane and water. The organic layer is dried with
Na.sub.2SO.sub.4 and the solvent removed in vacuo. The residue is
chromatographed (SiO.sub.2) with ethyl acetate to afford 95% of
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxyphenyl)methyl]benzimidazo-
l-2-yl}methyl)-N-(3-methylbutyl)carboxamide (Compound 1). Mass Spec
M.sup.+ 537.
EXAMPLE 3
General Procedure for the Preparation of benzimidazoles as Shown in
Scheme 3
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-({2-[3-(4-methylpip-
erazinyl)propoxy]phenyl}methyl)-benzimidazol-2-yl]methyl}carboxamide
[0120] 22
[0121] A solution of 5.0 g (30.0 mmole) of
2-(chloromethyl)benzimidazole in 25 mL of anhydrous
1-methyl-2-pyrrolidinone is treated at 0.degree. C. with 17.4 mL
(150 mmole) of isoamylamine. The reaction mixture is allowed to
warm to room temperature and stir for 16 hr. The reaction mixture
is poured into 800 mL of ice/water, and the tan solid filtered and
dried to afford 6.08 g (93%) of
(benzimidazol-2-ylmethyl)(3-methylbutyl)amine.
[0122] A solution of 3.0 g (13.8 mmole) of
(benzimidazol-2-ylmethyl) (3-methylbutyl)amine in 150 mL of 1:1:1
ethyl acetate, acetone, water is treated with 3.66 g (34.5 mmole)
Na.sub.2CO.sub.3 and a solution of 3.22 g (13.7 mmole) of
2-chloro-3,4-dimethoxybenzoyl chloride in 50 mL of acetone at
0.degree. C. The resulting mixture is allowed to warm to room
temperature for 1 hour. The reaction solution is dilluted with 300
mL of ethyl acetate and then washed with 2.times.60 mL water,
1.times.60 mL sat. NaHCO.sub.3, and 1.times.60 mL of brine. The
resulting organic layer is dried over anhydrous Na.sub.2SO.sub.4
and the solvent removed in vacuo. The residue is treated with 150
mL of methanol and 1.1 g of NaOH at reflux for 2 hours and then
allowed to cool to room temperature for 2 hours. The resulting
solution is evaporated under reduced pressure and partioned between
ethyl acetate 200 mL and water 100 mL. The ethyl acetate extracts
are dried over anhydrous Na.sub.2SO.sub.4 and the solvent removed
in vacuo. The resulting residue is flash chromatographed (1:1 ethyl
acetate/hexanes) to afford 2.82 g (49%) of
N-(benzimidazol-2-ylmethyl)(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbuty-
l)carboxamide.
[0123] A solution of 1.0 g (2.4 mmole) of
N-(benzimidazol-2-ylmethyl)(2-ch-
loro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-carboxamide and 1.56 g
(4.8 mmole) of Cs.sub.2CO.sub.3 in 5 mL anhydrous
N,N-dimethylformamide is treated with 0.64 g (2.88 mmole) of
2-(chloromethyl)phenyl methylsulfonate at room temperature and
heated to 50.degree. C. for 1 hr. The reaction is cooled to room
temperature, diluted with 60 mL of ethyl acetate and washed with
3.times.20 mL water and 1.times.20 mL brine, dried over anhydrous
Na.sub.2SO.sub.4 and the solvent is removed in vacuo. The resulting
residue is flash chromatographed (2% MeOH/CH.sub.2Cl.sub.2/0.5%
NH.sub.4OH) to afford 1.26 g (88%) of
2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carbonylamino]met-
hyl}benzimidazolyl)methyl]-phenyl methylsulfonate.
[0124] A solution of 1.2 g (2.0 mmole) of
2-[(2-{[(2-chloro-3,4-dimethoxyp-
henyl)-N-(3-methylbutyl)-carbonylamino]-methyl}benzimidazolyl)methyl]pheny-
l methylsulfonate and 0.321 g (8.0 mmole) of NaOH in methanol is
warmed to 50.degree. C. for 3.5 hours. The resulting mixture is
cooled to room temperature, evaporated at reduced pressure, diluted
with 100 mL of ethyl acetate, washed with 1.times.30 mL sat.
NH.sub.4Cl and 1.times.30 mL brine, dried over anhydrous
Na.sub.2SO.sub.4 and the solvent is removed in vacuo. The resulting
residue is flash chromatographed (3% MeOH/CH.sub.2Cl.sub.2/0.5%
NH.sub.4OH) to afford 0.864 g (83%) of
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-hydroxyphenyl)methyl]benzimidazo-
l-2-yl}methyl)-N-(3-methylbutyl) carboxamide.
[0125] A solution of 350 mg (0.67 mmole) of
(2-chloro-3,4-dimethoxyphenyl)- -N-({1-[(2-hydroxyphenyl)methyl]
benzimidazol-2-yl}methyl)-N-(3-methylbuty- l) carboxamide and 278
mg (2.01 mmole) of K.sub.2CO.sub.3 in 2 mL of anhydrous
N,N-dimethylformamide is treated with 79.1 .mu.l (0.737 mmole)
1-chloro-3-iodo propane at room temperature for 19 hr. The
resulting solution is diluted with 60 mL of ethyl acetate, washed
with 3.times.20 mL water and 1.times.20 mL of brine, dried over
anhydrous Na.sub.2SO.sub.4 and the solvent is removed in vacuo to
afford 400 mg (99%) of
(2-chloro-3,4-dimethoxyphenyl)-N-[(1-{[2-(3-chloropropoxy)phenyl-
]methyl}benzimidazol-2-yl)methyl]-N-(3-methylbutyl)
carboxamide.
[0126] A solution of 395 mg (0.66 mmole) of
(2-chloro-3,4-dimethoxyphenyl)-
-N-[(1-{[2-(3-chloropropoxy)phenyl}-methyl
benzimidazol-2-yl)methyl]-N-(3-- methylbutyl) carboxamide in 6 mL
of acetone is treated with 99 mg (3.30 mmole) of NaI at reflux for
16 hrs, at which time another 3.30 mmole of NaI is added and heated
for an additional 24 hrs. The solution is evaporated under reduced
pressure, the residue is diluted with 30 mL of ethyl acetate,
washed with 2.times.10 mL water and 1.times.10 mL brine, dried over
anhydrous Na.sub.2SO.sub.4 and the solvent is removed in vacuo. The
residue is dissolved in anhydrous 1-methyl-2-pyrrolidinone at 100
mg/ mL and 1 mL of the solution is treated with an excess of
N-methyl piperazine at 50.degree. C. for 16 hr. The resulting
solution is cooled to room temperature, washed with 3.times.1 mL
water and 1.times.1 mL brine, dried over anhydrous Na.sub.2SO.sub.4
and the solvent is removed in vacuo. The residue is prep-plate
chromatographed (5% MeOH/CH.sub.2Cl.sub.2/0.5% NH.sub.4OH) to
afford 65 mg (70%) of
2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-({2-[3-(4-methylpip-
erazinyl)propoxy]phenyl]methyl)benzimidazol-2-yl]methyl}carboxamide.
Mass Spec M.sup.+ 663.
[0127] Following the above procedures, compounds 11, 12, 15 and 16
are prepared starting from 2-(chloromethyl)imidazolo[5,4-b]pyridine
and 2-(chloromethyl)imidazolo[5,4-c]pyridine respectively. See also
Cleve et al., Justus Liebigs Ann. Chem. 1971, 747, 158-171.
EXAMPLE 4
[0128] The following compounds are prepared essentially according
to the procedures described in Examples 1-3, and as shown in
Schemes I-VII:
[0129] (a)
2-{2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carb-
onylamino]methyl}benzimidazolyl)methyl]phenoxy}acetic acid; Mass
Spec. M.sup.+ 581 amu.; (Compound 3).
[0130] (b)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[3-(2-pyrid-
ylmethyl)imidazolo[5,4-b]pyridin-2-yl]methyl}carboxamide; Mass Spec
M.sup.+ 509 amu.; (Compound 4).
[0131] (c)
2-(2-chlorophenyl)-N-({3-[(2-methoxyphenyl)methyl]imidazolo[5,4-
-b]pyridin-2-yl}methyl)-N-(3-methylbutyl)acetamide Mass Spec
M.sup.+ 492 amu.; (Compound 5).
[0132] (d)
[2-chloro-4-(methylethoxy)phenyl]-N-({3-[(2-methoxyphenyl)methy-
l]imidazolo[5,4-b]pyridin-2-yl}methyl)-N-(3-methylbutyl)carboxamide;
Mass Spec M.sup.+ 536 amu.; (Compound 6).
[0133] (e)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxyphenyl)methyl]i-
midazolo[4,5-c]pyridin-2-yl}methyl)-N-(3-methylbutyl)carboxamide;
Mass Spec M.sup.+ 538 amu.; (Compound 7).
[0134] (f)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-[(1-{[3-(3-p-
yrrolidinylpropoxy)phenyl]methyl}benzimidazol-2-yl)methyl]carboxamide;
Mass Spec M.sup.+ 634 amu.; (Compound 8).
[0135] (g)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-[(1-prop-2-e-
nylbenzimidazol-2-yl)methyl]carboxamide; Mass Spec M.sup.+ 457
amu.; (Compound 9).
[0136] (h)
2-(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carbonyl-
amino]methyl}-1-[(2-methoxyphenyl)methyl]benzimidazol-4-yloxy)acetic
acid; (Compound 10).
[0137] (i)
2-{2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carb-
onylamino]methyl}benzimidazolyl)methyl]phenoxy-N-(methylsulfonyl)
acetamide; (Compound 11).
[0138] (j)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-({1-[(2-(2H--
1,2,3,4-tetrazol-5-yl)phenyl)methyl]benzimidazol-2-yl}methylcarboxamide;
(Compound 12).
[0139] (k)
2-(2-chlorophenyl)-N-({1-[(2-chlorophenyl)methyl]benzimidazol-2-
-yl}methyl)-N-pentylacetamide; Mass Spec. M.sup.+ 495 amu.;
(Compound 13).
[0140] (l)
2-(2-chlorophenyl)-N-({1-[(2-chlorophenyl)methyl]benzimidazol-2-
-yl}methyl)-N-(3-methylbutyl)acetamide; Mass Spec. M.sup.+ 495
amu.; (Compound 14).
[0141] (m)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxy-5-nitrophenyl)-
methyl]benzimidazol-2-yl}methyl)-N-(3-methylbutyl)carboxamide; Mass
Spec. M.sup.+ 583 amu.; (Compound 15).
[0142] (n)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxyphenyl)methyl]b-
enzimidazol-2-yl}methyl)-N-pentylcarboxamide; Mass Spec. M.sup.+
537 amu.; (Compound 16).
[0143] (o)
N-({3-[(3,5-dichlorophenyl)methyl]imidazolo[5,4-b]pyridin-2-yl}-
methyl)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carboxamide; Mass
Spec. M.sup.+ 577 amu.; (Compound 17).
[0144] (p)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-hydroxyphenyl)methyl]b-
enzimidazol-2-yl}methyl)-N-(3-methylbutyl)carboxamide; Mass Spec.
M.sup.+ 523 amu.; (Compound 18).
[0145] (q)
2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carbony-
lamino]methyl}benzimidazolyl)methyl]phenyl methylsulfonate; Mass
Spec. M.sup.+ 601 amu.; (Compound 19).
[0146] (r)
(2-chloro-3,4-dimethoxyphenyl)-N-[(1-{[5-(hydroxyethyl)-2-metho-
xyphenyl]methyl}benzimidazol-2-yl)methyl]-N-(3-methylbutyl)
carboxamide; Mass Spec. M.sup.+ 581 amu.; (Compound 20).
[0147] (s)
N-({1-[(5-acetyl-2-methoxyphenyl)methyl]benzimidazol-2-yl}methy-
l)(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carboxamide; Mass
Spec. M.sup.+ 579 amu.; (Compound 21).
[0148] (t)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-chlorophenyl)methyl]be-
nzimidazol-2-yl}methyl)-N-(3-methylbutyl)carboxamide; Mass Spec.
M.sup.+ 541 amu.; (Compound 22).
[0149] (u)
(2-chloro-3,4-dimethoxyphenyl)-N-{[1-({3-[3-(methylamino)propox-
y]phenyl}methyl)benzimidazol-2-yl]methyl}-N-(3-methylbutyl)carboxamide;
Mass Spec. M.sup.+ 594 amu.; (Compound 23).
[0150] (v)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-({3-[3-(-
4-methylpiperazinyl)propoxy]phenyl}methyl)
benzimidazol-2-yl]methyl}carbox- amide; Mass Spec. M.sup.+ 663
amu.; (Compound 24).
[0151] (w)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-(2-pyrid-
ylmethyl)benzimidazol-2-yl]methyl}carboxamide; Mass Spec. M.sup.+
508 amu.; (Compound 25).
[0152] (x)
(2-chloro-3,4-dimethoxyphenyl)-N-{[1-({3-[2-(ethylamino)ethoxy]-
phenyl}methyl)benzimidazol-2-yl]methyl}-N-(3-methylbutyl)carboxamide;
Mass Spec. M.sup.+ 594 amu.; (Compound 26).
[0153] (y)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-fluorophenyl)methyl]be-
nzimidazol-2-yl}methyl)-N-(3-methylbutyl)carboxamide; Mass Spec.
M.sup.+ 525 amu.; (Compound 27).
[0154] (z)
N-butyl(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(2-methoxyphenyl)m-
ethyl]benzimidazol-2-yl}methyl)carboxamide; Mass Spec. M.sup.+ 523
amu.; (Compound 28).
[0155] (aa)
(2-chloro-3,4-dimethoxyphenyl)-N-({3-[(3-chlorophenyl)methyl]i-
midazolo[5,4-b]pyridin-2-yl}methyl)-N-(3-methylbutyl)carboxamide;
Mass Spec. M.sup.+ 542 amu.; (Compound 29).
[0156] (bb)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-({1-[(2-met-
hylphenyl)methyl]benzimidazol-2-yl}methyl)carboxamide; Mass Spec.
M.sup.+ 521 amu.; (Compound 30).
[0157] (cc)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-[(1-{[3-(3--
morpholin-4-ylpropoxy)phenyl]methyl}benzimidazol-2-yl)methyl]carboxamide;
Mass Spec. M.sup.+ 650 amu.; (Compound 31).
[0158] (dd)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-({3-[2--
(4-methylpiperazinyl)ethoxy]phenyl}methyl)-benzimidazol-2-yl]methyl}carbox-
amide; Mass Spec. M.sup.+ 649 amu.; (Compound 32).
[0159] (ee)
3-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carbon-
ylamino]methyl}benzimidazolyl)methyl]phenyl methylsulfonate; Mass
Spec. M.sup.+ 601 amu.; (Compound 33).
[0160] (ff)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-[(1-{[3-(2--
piperidylethoxy)phenyl]methyl}benzimidazol-2-yl)methyl]carboxamide;
Mass Spec. M.sup.+ 634 amu.; (Compound 34).
[0161] (gg)
(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(3-hydroxyphenyl)methyl]-
benzimidazol-2-yl}methyl)-N-(3-methylbutyl) carboxamide; Mass Spec.
M.sup.+ 523 amu.; (Compound 35).
[0162] (hh) ethyl
2-{2-[(2-{[(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbut-
yl)carbonylamino]methyl}benzimidazolyl) methyl]-phenoxy)acetate;
Mass spec. M.sup.+ 609 amu.; (Compound 36).
[0163] (ii)
(2-chloro-3,4-dimethoxyphenyl)-N-({3-[2-methoxyphenyl)methyl]i-
midazolo[5,4-b]pyridin-2-yl}methyl)-N-(3-methylbutyl) carboxamide;
Mass Spec. M.sup.+ 5 38 amu.; (Compound 37).
[0164] (jj)
N-({1-[(3-amino-6-methoxyphenyl)methyl]benzimidazol-2-yl}methy-
l)(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)carboxamide; Mass
Spec. M.sup.+ 552 amu.; (Compound 38).
[0165] (kk)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-{[1-({2-[2--
(4-methylpiperazinyl)ethoxy]phenyl}methyl)-benzimidazol-2-yl]methyl}carbox-
amide; Mass Spec. M.sup.+ 649 amu.; (Compound 39).
[0166] (11)
N-butyl(2-chloro-3,4-dimethoxyphenyl)-N-({1-[(3-fluorophenyl)m-
ethyl]benzimidazol-2-yl}methyl)carboxamide; Mass Spec. M.sup.+ 511
amu.; (Compound 40).
[0167] (mm)
(2-chloro-3,4-dimethoxyphenyl)-N-(3-methylbutyl)-N-[(1-{[2-(tr-
ifluoromethyl)phenyl]methyl}benzimidazol-2-yl)methyl]carboxamide;
Mass Spec. M.sup.+ 575 amu.; (Compound 41).
[0168] (nn)
[2-chloro-4-(methylethoxy)phenyl]-N-(3-methylbutyl)-N-({1-[(2--
nitrophenyl)methyl]benzimidazol-2-yl}methylcarboxamide; Mass Spec.
M.sup.+ 550 amu.; (Compound 42).
[0169] (oo)
(2-chloro-3,4-dimethoxyphenyl)-N-[(4-methyoxy-1-prop-2-enylben-
zimidazol-2-yl)methyl]-N-(3-methylbutyl)carboxamide; Mass Spec.
M.sup.+ 487 amu.; (Compound 43).
EXAMPLE 5
Ligand Binding Assay on Sf9 Cell Membranes Expressing the BK-2
Receptor
[0170] This assay is a standard assay of BK-2 receptor binding, and
is used to determine the high affinity of compounds of this
invention for the BK-2 (bradykinin B.sub.2) receptor.
[0171] Binding Buffer: 50 mM Tris, pH 7.0 (4.degree. C.), 0.14
grams per liter bacitracin (approx. 50,000 units of activity/liter,
lot# 103746 from Amersham), and 10.sup.-6 M captopril. Captopril is
purchased from Sigma C-4042, 2.17 mg in 10 ml of milli-Q water
produces a 10.sup.-3 M stock. Stock can be stored for 3 weeks in
the refrigerator. 1.0 ml of stock per liter buffer=10.sup.-6 M
final concentration.
[0172] Ligand Preparation: 0.25 nM .sup.3H-Bradykinin is used. 10
.mu.l of stock+100 ml of binding buffer gives approximately 600
cpm/5 ul aliquot.
[0173] Non-Specific Preparation: NS binding is defined by unlabeled
bradykinin at 1 .mu.M final concentration. Aliquots are stored at
-20.degree. C. in 0.5% BSA at a concentration of 10.sup.-3 M.
Aliquots are then diluted 1:100 for an intermediate concentration
of 10.sup.-5 M.
[0174] Baculovirus-infected Sf9 cells expressing recombinant human
bradykinin B.sub.2 receptors are harvested 48 hours post infection
via centrifugation at 3000.times.g. Cells are washed with ice-cold
PBS and stored at -70.degree. C. until needed. Frozen cell pellets
are resuspended in ice cold Washing Buffer (5 mM Tris pH 7.0) and
homogenized via POLYTRON for 30 seconds at setting 5. Membranes are
centrifuged at 40,000.times.g for 10 min. Pellets are resuspended
in Washing Buffer with the aid of a polytron and centrifuged again.
Membranes are resuspended in binding buffer at a concentration of
133 .mu.g/ml. This corresponds to 20 .mu.g of protein per 150
.mu.l.
[0175] When measuring non-specific binding, incubations contain 150
.mu.l of Sf9 cell membranes prepared as described above, 50 .mu.l
.sup.3H-Bradykinin (0.25 nM), 25 .mu.l unlabeled bradykinin at 1
.mu.M final concentration and 2 .mu.l DMSO. Incubations for
determining test compound binding contain 175 .mu.l of Sf9 cell
membranes, 50 .mu.l .sup.3H-Bradykinin (0.25 nM), and test compound
in 2 .mu.l DMSO. The concentration of the test compound is
generally 1 .mu.M for displacement studies. The binding reaction
components are incubated for 2 hrs at 4.degree. C. in Falcon U
bottom plates. Plates are harvested on the microbeta harvester onto
0.5% PEI pretreated unifilters. After harvesting, the filters are
dried overnight. 17 .mu.l of beta-scint is added to each well
before the unifilters are counted in the microbeta counters. Data
are collected in duplicate determinations, averaged and %
inhibition of total specific binding is calculated. Total Specific
Binding=Total-Nonspecific. In some cases, the amounts of unlabeled
drug are varied and total displacement curves of binding are
carried out. Data are converted to a form for the calculation of
IC.sub.50 and Hill Coefficient (nH). Ki's are subsequently
determined by the Cheng-Prusoff equation (Cheng, Y. C.; Prusoff,
W.C. Biochem. Pharmacol. 1972, 22, 3099-3108). In the described
assay, compounds of the invention have Ki's of less than 1 uM,
preferred compounds of the invention exhibit Ki values of less than
500 nM and more preferred compounds of the invention exhibit Ki
values of less than 100 nM.
EXAMPLE 6
BK-2 Receptor Mediated Calcium Mobilization
[0176] The agonist and antagonist properties of the compounds of
the invention can be evaluated by the following assay.
[0177] CHO cells stably expressing the BK-2 receptor are grown in
Ham's F-12 media supplemented with 250 .mu.g/ml G418, 1 .mu.g/ml
tetracycline, 7 .mu.g/ml puromycin, 10% fetal bovine serum and 25
mM Hepes, pH=7.4. Forty-eight hours prior to assay, the cell growth
media is replaced with another medium that does not contain the
tetracycline. Twenty-four hours prior to experiment sodium butyrate
is added to a final concentration of 10 mM. On the day of assay,
cells, grown to 70-90% confluence in 96-well plates, are washed
with Krebs-Ringer buffer (25 mM HEPES, 5 mM KCl, 0.96 mM
NaH.sub.2PO.sub.4, 1 mM MgSO.sub.4, 2 mM CaCl.sub.2, 5 mM glucose,
and 1 mM probenecid, pH 7.4) and are then incubated for 1-2 hours
in the above buffer supplemented with Fluo3-AM (2.5 10 (g/ml;
Teflabs) at 37.degree. C. in an environment containing 5% CO.sub.2.
The wells are then washed twice with Krebs-Ringers buffer.
Agonist-induced (bradykinin) calcium mobilization is monitored
using either Fluoroskan Ascent (Labsystems) or FLIPR (Molecular
Devices) instruments. The agonists, either bradykinin or drug
candidates, are added to the cells and fluorescence responses are
continuously recorded for up to 5 min. For the examination of
antagonist drug candidates, compounds, at a concentration of 1 uM
in DMSO, are preincubated with the cells for up to 30 minutes prior
to administration of the bradykinin agonist. Bradykinin agonist is
generally applied at a concentration suffiencient to induce 50%
maximal activity. Responses are recorded for up to 5 min.
Kaleidagraph software (Synergy Software, Reading, Pa.) is utilized
to fit the data to the equation y=a*(1/(1+(b/x)c)) to determine the
EC.sub.50 value or IC.sub.50 value for the response. In this
equation, y is the maximum fluorescence signal, x is the
concentration of the agonist or antagonist, a is the Emax, b
corresponds to the EC.sub.50 or IC.sub.50 value, and, finally, c is
the Hill coefficient.
EXAMPLE 7
Preparation of Radiolabeled Probe Compounds of the IInvention
[0178] The compounds of the invention are prepared as radiolabeled
probes by carrying out their synthesis using precursors comprising
at least one atom that is a radioisotope. The radioisotope is
preferably selected from of at least one of carbon (preferably
.sup.14C), hydrogen (preferably .sup.3H), sulfur (preferably
.sup.35S), or iodine (preferably .sup.125I). Such radiolabeled
probes are conveniently synthesized by a radioisotope supplier
specializing in custom synthesis of radiolabeled probe compounds.
Such suppliers include Amersham Corporation, Arlington Heights,
Ill.; Cambridge Isotope Laboratories, Inc. Andover, Mass.; SRI
International, Menlo Park, Calif.; Wizard Laboratories, West
Sacramento, Calif.; ChemSyn Laboratories, Lexena, Kans.; American
Radiolabeled Chemicals, Inc., St. Louis, Mo.; and Moravek
Biochemicals Inc., Brea, Calif.
[0179] Tritium labeled probe compounds are also conveniently
prepared catalytically via platinum-catalyzed exchange in tritiated
acetic acid, acid-catalyzed exchange in tritiated trifluoroacetic
acid, or heterogeneous-catalyzed exchange with tritium gas. Such
preparations are also conveniently carried out as a custom
radiolabeling by any of the suppliers listed in the preceding
paragraph using the compound of the invention as substrate. In
addition, certain precursors may be subjected to tritium-halogen
exchange with tritium gas, tritium gas reduction of unsaturated
bonds, or reduction using sodium borotritide, as appropriate.
EXAMPLE 8
Use of Compounds of the Invention as Probes for BK-2 Receptors in
Cultured Cells and Tissue Samples
[0180] The presence of BK-2 receptors in cultured cells or tissue
samples may be ascertained by the procedures described by Hall and
Morton in the chapter entitled "Immunopharmacology of the
Bradykinin Receptor" of The Handbook of Immunopharmacology--The
Kinin Systems (1997) Academic Press, S. C. Farmer, editor, using
radiolabeled compounds of the invention prepared as described in
the preceding Example 7.
[0181] The invention and the manner and process of making and using
it, are now described in such full, clear, concise and exact terms
as to enable any person skilled in the art to which it pertains, to
make and use the same. It is to be understood that the foregoing
describes preferred embodiments of the present invention and that
modifications may be made therein without departing from the spirit
or scope of the present invention as set forth in the claims. To
particularly point out and distinctly claim the subject matter
regarded as invention, the following claims conclude this
specification.
* * * * *